We have developed a procedure for isolating DNA-dependent RNA polymerase from rooster liver nuclei that involves treatment of nuclei with pancreatic DNAse I to partially solubilize chromatin. The enzyme thus obtained is completely dependent on the addition of exogenous DNA for activity. We have developed a technique for identifying RNA polymerase II in nuclear lysates by binding 3H-alpha-amanitin to the polymerase and subsequently subjecting the proteins to electrophoresis in polyacrylamide gels under non-denaturing conditions. By this method we have identified two alpha-amanitin binding proteins, tentatively identified as two forms of RNA polymerase II.